Glutathione-coated nanoparticles for delivery of MKT-077 across the blood-brain barrier

ABSTRACT

Nanoparticle based MKT formulation. MKT is encapsulated by poly(ethylene glycol)ylated (PEGylated) poly-(lactide-co-glycolide) (PLGA) to form nanoparticles (NPs). To induce trans-BBB permeability, glutathione (GSH) is coated on the resulting NPs. Newly generated MKT-NPs showed BBB permeability and tau reduction in experimental models. Specifically, brain-targeting MKT NPs were developed with a glutathione coating, characterized, and shown to permeate BBB permeation insert models as a therapeutic for Alzheimer&#39;s disease and related tauopathies.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.15/151,973, filed May 11, 2016, which claims the benefit of U.S.Provisional Application Ser. No. 62/164,291, filed May 20, 2015, each ofwhich is hereby incorporated by reference herein in its entirety,including any figures, tables, nucleic acid sequences, amino acidsequences, or drawings.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates, generally, to therapies for tauopathies andcancer. More specifically, it relates to delivery of MKT-077nanoparticles across the blood-brain barrier for the treatment of suchdiseases and disorders.

2. Brief Description of the Prior Art

Alzheimer's disease (AD) is one of the most common causes of dementiaand death in elderly populations. As average lifespan continues toincrease, so does the prevalence of the disease. AD is characterized ascognitive impairment and disturbances that impact memory and behavior.AD is caused by neuronal inactivity and degeneration and leading toalterations in individuals' memory, thinking, speech, and behavior.Current estimates at the number of Americans currently suffering from ADplace the figure at 5.2 million, incidence of which is sure to rise withthe growth in the aging baby boomer generation, predicted to add anadditional 10 million AD patients in the coming future. Currently, AD isthe fifth most common cause of death in Americans over the age of 65 andthe sixth most common leading cause of death in America [7]. It isimperative that new forms of therapies targeted to AD are developed tocombat the morbidity of the disease. However, therapeutic interventionin AD is limited by the blood-brain barrier (BBB), which not onlyprotects the brain by limiting the permeation of potential toxins intoneural tissue but also by blocks certain drugs aimed at neurologicaldisorders.

Tau, the aggregation of which indicates the presence of AD, belongs tothe family of microtubule-associated proteins (MAP) and is anintrinsically disordered protein, lending it a number of potentialcellular functionalities including regulation of vesicle transport andcell signaling [1-3]. Aggregation and conformational changes of tauprotein render it unable to properly bind axonal microtubules in AD,leading to characteristic AD pathology [1, 4, 5]. Aberrant activity oftau leads to aggregation of tau protein into neurofibrillary bundles,causing toxicity and neurodegeneration in AD patients [6].

MKT-077 (MKT) is one such drug that has previously been shown to exhibitanti-tau and anti-cancer activities in cellular models. MKT is a highlywater soluble cationic rhodacyanine dye [8, 9]. Biochemical analysis ofMKT activity show that it selectively binds heat shock protein 70(Hsp70) in cells, a mediator of tau protein activity that prevents itsaccumulation by promoting binding to tubulin microtubules [10, 11]. Theinteractions of MKT with Hsp70 make this drug an interesting target forcancer therapies based on its anti-cancer effects on melanoma andcarcinoma of colon, breast, and pancreas [8].

However, phase 1 clinical trials of MKT in chemo-resistant solid tumorsrevealed irreversible renal toxicity in rat, dog, and human subjects [9,12].

Furthermore, despite MKT showing promise in reducing AD-relatedpathology in cellular models, it was shown that MKT has limited-to-nobrain permeation due to blockage by the BBB [13]. The BBB is formed bythe interaction of endothelial cells, astrocytes, and pericytes inneural tissue, create tight junctions that prevent toxins permeatinginto the brain by acting as a barrier. However, this same barrierinhibits drug delivery to alleviate AD's symptoms. For drugs to beeffective against AD, it is essential that they are able to surmount theBBB, an obstacle to brain drug delivery that emerges from theinteractions of endothelial cells with astrocytes and pericytes, whichblocks the permeation of potential toxins into neural tissue [14]. As aresult, a number of drug compounds are also unable to directly targetthe brain due to the BBB.

Attempts have been made to eliminate toxicity and traverse the BBB. Forexample, U.S. Pat. Nos. 8,067,370 and 8,569,239 to Wang et al. describesa biological delivery system comprising a carrier or an active compound,and a glutathione ligand or a glutathione derivative ligand. U.S. Pat.No. 8,003,128 to Kreuter et al. discusses a method of preparing a drugtargeting system for administering a pharmacologically active substanceto the CNS of a mammal across the BBB of the mammal using PLGA. U.S.Pat. No. 8,628,801 to Garreta teaches oral PEGylated nanoparticles forcarrying biologically active molecules such as anti-tumor agents. U.S.Pat. No. 8,603,501 teaches pharmaceutical compositions includingtarget-specific stealth nanoparticles useful in the treatment of cancerthat includes the anti-cancer agent and a diblock copolymer of PEG andPLA or PLGA. U.S. Patent Application Publication No. 2007/0148074 toSadoqi et al. describes polymer nanoparticles, for example includingPLGA, to entrap fluorescent dyes and increase their stability in vitroand in vivo.

PCT Application Publication No. WO 2012/106713 to Basilion et al.teaches targeted nanoparticle conjugates including a polyethyleneglycolylated nanoparticle, a hydrophobic therapeutic agent (e.g.,anti-cancer agent) coupled to the surface of the nanoparticle, and atargeting moiety coupled to polyethylene glycol of the nanoparticle fortargeting the composition to a cell associated with a disorder. PCTApplication Publication No. WO 2008/128123 to Brader et al. discussesactivated polymeric nanoparticle for targeted drug delivery, including abiocompatible polymer (e.g., PLGA), an amphiphilic stabilizing agent, anelectrophile that selectively reacts with a targeting agent and placesthe targeting agent on a biodegradable nanoshell loaded on an activeagent.

Koopaei, Mona N., Mohammad R. Khoshayand, Seyed H. Mostafavi, MohsenAmini, Mohammad R. Khorramizadeh, Mahmood J. Tehrani, Fatemeh Atyabi,and Rassoul Dinarvand. “Docetaxel Loaded PEG-PLGA Nanoparticles:Optimized Drug Loading, In-vitro Cytotoxicity and In-vivo AntitumorEffect.” Iranian Journal of Pharmaceutical Researches 13.3 (2014):819-33. PubMed. Web. 6 Nov. 2014 describes docetaxel loaded PEG-PLGAnanoparticles as anticancer agents. Avgoustakis, Konstantinos.“Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles:Preparation, Properties and Possible Applications in Drug Delivery.”Current Drug Delivery 1.4 (2004): 321-33 teaches the preparation,properties and potential applications in drug delivery of PLGA-PEGnanoparticles. Sankar, Renu, and Vilwanathan Ravikumar.“Biocompatibility and Biodistribution of Suberoylanilide Hydroxamic AcidLoaded Poly (DL-lactide-co-glycolide) Nanoparticles for Targeted DrugDelivery in Cancer.” Biomed Pharmacother. (2014), PubMed discussessuberoylanilide hydroxamic acid (SAHA) loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles used to carry variouschemotherapy agents into cancer cells for targeted drug delivery.

However, none of the foregoing references have been able to providenon-toxic nanoparticles and delivery system of MKT-077 that is alsocapable of successfully crossing the BBB. Accordingly, what is needed isa therapy and nanoparticle formulation that is capable of overcoming theforegoing toxicity and BBB permeability issues. However, in view of theart considered as a whole at the time the present invention was made, itwas not obvious to those of ordinary skill in the field of thisinvention how the shortcomings of the prior art could be overcome.

While certain aspects of conventional technologies have been discussedto facilitate disclosure of the invention, Applicants in no way disclaimthese technical aspects, and it is contemplated that the claimedinvention may encompass one or more of the conventional technicalaspects discussed herein.

The present invention may address one or more of the problems anddeficiencies of the prior art discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

In this specification, where a document, act or item of knowledge isreferred to or discussed, this reference or discussion is not anadmission that the document, act or item of knowledge or any combinationthereof was at the priority date, publicly available, known to thepublic, part of common general knowledge, or otherwise constitutes priorart under the applicable statutory provisions; or is known to berelevant to an attempt to solve any problem with which thisspecification is concerned.

BRIEF SUMMARY OF THE INVENTION

The long-standing but heretofore unfulfilled need for brain-targetingtherapies for tauopathies is now met by a new, useful, and nonobviousinvention.

In an embodiment, the current invention is a composition itself or amethod of treating a tauopathy or a cancer by administering atherapeutically effective amount of such composition. The compositioncomprises MKT admixed with a biocompatible, non-toxic polymer to formnanoparticles (e.g., average size of less than 300 nm) with MKTencapsulated therewithin. The nanoparticles each have a surface coatedby glutathione (e.g., 2% w/v), such that the hydrophilic nature ofglutathione allows MKT to effective permeate across the BBB despite thetight junctions of the BBB increasing their expression (duringadministration of the composition) to decrease toxicity that can crossthe BBB.

The biocompatible, non-toxic polymer may include poly(ethyleneglycol)ylated poly-(lactide-co-glycolide) or other suitable hydrophilicpolymer to encapsulate the MKT, wherein this hydrophilicity facilitatesan interaction of the glutathione with transporters present on the BBB.

MKT can have a sustained release from the nanoparticles of over about 72hours, and during this time, about 100% of the MKT would be releasedfrom the nanoparticles.

The composition may further include a plurality of poly(ethylene glycol)end groups around the surface of each nanoparticle, where the end groupscan be longer or otherwise extend further away from the nanoparticlesurface than the glutathione that is disposed on the nanoparticlesurface. This provides a surface area of the end grounds to interactwith transporters present on the BBB.

These and other important objects, advantages, and features of theinvention will become clear as this disclosure proceeds.

The invention accordingly comprises the features of construction,combination of elements, and arrangement of parts that will beexemplified in the disclosure set forth hereinafter and the scope of theinvention will be indicated in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a schematic of formulated NPs. MKT drug is encapsulated in thehollow core of the NP sphere that is created as PEG-PLGA lipid chainscoalesce upon evaporation of the oil phase during formulation. PEG endgroups help the NPs evade the RES system and maintain hydrophilicity inblood. GSH is coated onto the NPs surface due to electrostaticinteractions of charged functional groups on both GSH and PEG-PLGAvector. 178×142 mm (72×72 DPI)

FIG. 2A is an SEM visualization of blank NPs. Samples were dilutedaccording to instrument specifications and visualization was donethrough JOEL JSM-6490LV (JOEL Industries, Tokyo, Japan). Samples wereread at 65,000× magnification and 4 kV acceleration voltage for blankNPs and MKT NPs and 8 kV acceleration voltage for MKT Glu-NPs samples.267×67 mm (72×72 DPI)

FIG. 2B is an SEM visualization of MKT NPs. Samples were dilutedaccording to instrument specifications and visualization was donethrough JOEL JSM-6490LV (JOEL Industries, Tokyo, Japan). Samples wereread at 65,000× magnification and 4 kV acceleration voltage for blankNPs and MKT NPs and 8 kV acceleration voltage for MKT Glu-NPs samples.267×67 mm (72×72 DPI)

FIG. 2C is an SEM visualization of MKT Glu-NPs. Reflective coating onsurface of MKT Glu-NPs suggests the successful coating of GSH onto theNPs formulation. Samples were diluted according to instrumentspecifications and visualization was done through JOEL JSM-6490LV (JOELIndustries, Tokyo, Japan). Samples were read at 65,000× magnificationand 4 kV acceleration voltage for blank NPs and MKT NPs and 8 kVacceleration voltage for MKT Glu-NPs samples. 267×67 mm (72×72 DPI)

FIG. 3 is a graphical illustration showing in vitro drug release data ofdrug solution, MKT NPs, and MKT Glu-NPs over a 7-day study period. MKTGlu-NPs exhibited sustained drug release up to 72 hours until fullcontent of drug was released from NP vector, and MKT NPs showedsustained drug release up to 7 days. Samples were read through UVspectroscopy at wavelength of 492 nm (λ_(max)) in triplicate (n=3) andreported as mean percentage drug release±SD. 242×74 mm (72×72 DPI)

FIG. 4A is a graphical illustration showing trans-BBB brain permeationof drug solution vs. MKT Glu-NPs, obtained through TRANSWELL in vitroBBB model established through the co-culture of RBE4 (rat brainendothelial) cells with C6 (rat astrocytoma) cells. MKT Glu-NPs showedthe greatest permeation through the BBB model over the 48-hour studyperiod. Treatments were carried out in quadruplicate (n=4) and readthrough UV spectroscopy at 492 nm (λ_(max)) for drug content. Values arereported as mean percent drug permeated±SD. 238×75 mm (72×72 DPI)

FIG. 4B is a graphical illustration showing trans-BBB brain permeationof drug solution vs. MKT NPs, obtained through TRANSWELL in vitro BBBmodel established through the co-culture of RBE4 (rat brain endothelial)cells with C6 (rat astrocytoma) cells. MKT Glu-NPs showed the greatestpermeation through the BBB model over the 48-hour study period.Treatments were carried out in quadruplicate (n=4) and read through UVspectroscopy at 492 nm (λ_(max)) for drug content. Values are reportedas mean percent drug permeated±SD. 238×75 mm (72×72 DPI)

FIG. 5A illustrates MKT nanoparticles decreasing tau levels in acellular model. HeLa cells stably transfected with human tau weretreated for 24 hours with uncoated/Unc np (Vehicle − or MKT+) orsolution (Vehicle − or MKT+) or GSH np (Vehicle − or MKT +).

FIG. 5B is a quantification plot of the Western blot (see FIG. 5A) afterGAPDH normalization and comparison with respective vehicle (veh) controlshow reduction in tau level by MKT. Replicate blots were used for theanalysis. 73×115 mm (150×150 DPI)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of the preferred embodiments,reference is made to the accompanying drawings, which form a partthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contentclearly dictates otherwise. As used in this specification and theappended claims, the term “or” is generally employed in its senseincluding “and/or” unless the context clearly dictates otherwise.

As discussed, MKT is a cationic rhodacyanine dye shown to interact withHsp70 to regulate tau protein. MKT has shown anti-cancer effects onmelanoma and carcinoma of colon, breast, and pancreas as well. However,phase 1 clinical trials of MKT in chemo-resistant solid tumors revealedirreversible renal toxicity in rat, dog, and human subjects.Furthermore, it was shown that MKT does not cross the BBB. As such, ananoparticle based formulation was developed herein to overcome theseparticular toxicity and BBB permeability issues. In an embodiment,poly(ethylene glycol)ylated (PEGylated) PLGA nanoparticles (NPs) wereused to encapsulate MKT, though other suitable encapsulation methods arecontemplated herein as well. To induce trans-BBB permeability,glutathione (GSH) was coated on the resulting NPs, though other suitablehydrophilic molecules or formulations can be used as well for coatingthe NPs. Examples of alternatives to GSH include, but are not limitedto, transport systems based on thiamine (or other amino acids) andtransferrin for effective BBB permeation [33].

Though PEGylated PLGA is well known to be safe, biocompatible, non-toxicand restorable through natural pathways, other encapsulating polymersand formulations are contemplated herein as well for preparation of theNPs. Examples include, but are not limited to, lipids or oils, gelatin,sodium alginate, gum arabic, starch, tragacanth, shellac, paraffin wax,polylactic acid (PLA), polycaprolactone (PCL), methyl cellulose, pectin,carrageenan, alginates, methyl cellulose, casein, bovine albumin serum,chitosan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethylcellulose, cellulose acetate phthalate, carmellose, polyvinyl alcohol,polystyrene, polyurethane, polyvinylpyrrolidone, polymethacrylate,polyvinyl acetate, polyhydroxyethyl methacrylate, polyvinyl chloride,polyacrylate, polyacrylamide, polyethylene glycol, polyester, polyurea,and polyamide, among other suitable polymers and formulations that maybe used to prepare the NPs.

Examples of lipids that may be used include, but are not limited to,derivatives of glycerophospholipids, glycerolipids, sphingolipids,sterols, fatty acyl amides, prenols, ceramides, cholesterols, lecithin,glyceryl behenate (COMPRITOL), glyceryl palmitostearate (PRECIROL),glycerol monosterol (MONO STEROL), glycerol disterate, sulfatides,phosphosphingolipids, phosphatidylcholines, phosphatidic acids,phosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines,and phosphor lipids, among other suitable lipids that may be used toprepare multilayered nanostructures.

Examples of oils that may be used include, but are not limited to,safflower oil, sesame oil, corn oil, castor oil, coconut oil, almondoil, cotton seed oil, soybean oil, olive oil, mineral oil, spearmintoil, clove oil, lemon oil, peppermint oil, triacetin, tributryin, ethylbutyrate, ethyl caprylateoleic acid, ethyl oleate, isopropyl myristateand ethyl caprylate, among other suitable oils that may be used toprepare multilayered nanostructures.

The term “administration” and variants thereof (e.g., “administering” acompound) in reference to a compound of the invention means introducingthe compound or a prodrug of the compound into the system of the animalin need of treatment. When a compound of the invention or prodrugthereof is provided in combination with one or more other active agents(e.g., a cytotoxic agent, etc.), “administration” and its variants areeach understood to include concurrent and sequential introduction of thecompound or prodrug thereof and other agents. Administration can beaccomplished in number of ways including, but not limited to, parenteral(such term referring to intravenous and intra-arterial as well as otherappropriate parenteral routes), subcutaneous, peritoneal, inhalation,vaginal, rectal, nasal, or instillation into body compartments.

Formulations suitable for parenteral administration include, forexample, aqueous sterile injection solutions, which may containantioxidants, buffers, bacteriostats, and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and nonaqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze dried (lyophilized) conditionrequiring only the condition of the sterile liquid carrier, for example,water for injections, prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powder, granules, tablets,etc. It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations of the subject inventioncan include other agents conventional in the art having regard to thetype of formulation in question. The pharmaceutical composition can beadapted for various forms of administration. Administration can becontinuous or at distinct intervals as can be determined by a personskilled in the art, unless otherwise noted.

Administration will often depend upon the amount of compoundadministered, the number of doses, and duration of treatment. In anembodiment, multiple doses of the agent are administered. The frequencyof administration of the agent can vary depending on any of a variety offactors, such as extent of tau protein levels, tumor volume, and thelike. The duration of administration of the agent, e.g., the period oftime over which the agent is administered, can vary, depending on any ofa variety of factors, including patient response, etc.

The amount of the agent contacted (e.g., administered) can varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry, and the like. Detectablyeffective amounts of the agent of the present disclosure can also varyaccording to instrument and film-related factors. Optimization of suchfactors is well within the level of skill in the art, unless otherwisenoted.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients, in the specified amounts,as well as any product which results, directly or indirectly, fromcombination of the specified ingredients in the specified amounts.Generally, the specified ingredients, or pharmaceutically acceptablesalts and derivatives thereof, are suitable agents for use in thediagnosis, mitigation, treatment, cure, or prevention of disease in asubject, specifically but not exclusively effective in the treatmentand/or prevention of estrogen receptor-mediated disorders, includingspecifically the treatment and/or prevention of breast cancer, whenadministered in an effective amount to a subject in need thereof.

As used herein, the terms “nanostructure” and “nanoparticle” may be usedinterchangeably to refer to any polymeric micelle, lipid micelle, hybridlipid-polymer micelle, liposome, niosomes, transferosome,liponanoparticle, lipid nanoparticles, nanostructured lipid nanocarriers(NLC), solid lipid nanoparticles (SLN), hybrid lipid-polymernanostructures, bicelle, polymerosomes, lamellar structures, and lipidvesicles, among other delivery systems that can be used suitably todeliver one or more active pharmaceutical agent(s).

A combination of polymers and/or lipids can be used to prepare thenanoparticles. The nanoparticles can be prepared using electrostaticinteraction, self-assembly, ionotropic gelation, cross-linking,coacervation, homogenization-solvent evaporation, sonication,ultrasound, nanoprecipitation, spray drying, high pressurehomogenization, layer by layer, freeze drying, hot-melt homogenization,film formation, co-solvent evaporation, high pressure instruments suchas NANODEBEE, and coating or solvent emulsion methods, alone or incombination.

As used herein, the term “neoproliferative disease” means a neoplasm,cancer, or precancerous lesion. The neoplasm or cancer may be benign ormalignant.

As used herein, the term “subject,” “patient,” or “organism” includeshumans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses).Typical hosts to which an agent(s) of the present disclosure may beadministered will be mammals, particularly primates, especially humans.For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.

The pharmaceutical compositions of the subject invention can beformulated according to known methods for preparing pharmaceuticallyuseful compositions. Furthermore, as used herein, a “pharmaceuticallyacceptable excipient,” “pharmaceutically acceptable diluent,”“pharmaceutically acceptable carrier,” or “pharmaceutically acceptableadjuvant” means any of the standard pharmaceutically acceptablecarriers, such as a solvent, suspending agent or vehicle, for deliveringthe compound or compounds in question to the mammal. The carrier may beliquid or solid and is selected with the planned manner ofadministration in mind. The pharmaceutically acceptable carrier caninclude diluents, adjuvants, and vehicles, as well as implant carriers,and inert, non-toxic solid or liquid fillers, diluents, or encapsulatingmaterial that does not react with the active ingredients of theinvention. Examples include, but are not limited to, phosphate bufferedsaline, liposomes, physiological saline, water, and emulsions, such asoil/water emulsions. The carrier can be a solvent or dispersing mediumcontaining, for example, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils. The carrier can also include anyand all other vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. Formulations aredescribed in a number of sources that are well known and readilyavailable to those skilled in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. For example,Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations which can beused in connection with the subject invention. “A pharmaceuticallyacceptable excipient, diluent, carrier and/or adjuvant” as used in thespecification and claims includes one or more such excipients, diluents,carriers, and adjuvants.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

As used herein, the term “precancerous” refers to cells or tissues thathave characteristics relating to changes that may lead to malignancy orcancer, such as mutations controlling cell growth and proliferation.Examples include adenomatous growths in breast and prostate tissue, orfor example, conditions of dysplastic nevus syndromes, polyposissyndromes, prostatic dysplasia, and other neoplasms, whether clinicallyidentifiable or not.

The term “prevention” is used herein to refer to the management of thefactors that could lead to disease or disorder, so as to prevent theoccurrence of the disease or disorder. Specifically, the disease ordisorder includes, but is not limited to, various tauopathies andcancers.

As used herein, the terms “prophylactically treat” or “prophylacticallytreating” refers completely or partially preventing (e.g., about 50% ormore, about 60% or more, about 70% or more, about 80% or more, about 90%or more, about 95% or more, or about 99% or more) a condition or symptomthereof and/or may be therapeutic in terms of a partial or complete cureor alleviation for a condition and/or adverse effect attributable to thecondition.

A “safe and effective amount” refers to the quantity of a component orcomposition that is sufficient to yield a desired therapeutic responsewithout undue adverse side effects (such as toxicity, irritation, orallergic response) commensurate with a reasonable benefit/risk ratiowhen used in the manner of this invention for the treatment and/orprevention of tauopathies and cancer.

The term “therapeutically effective amount” as used herein means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue, system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician. In reference to tauopathies, an effective amountcomprises an amount sufficient to cause a reduction in tau proteinlevels or a reduction in growth of tau protein levels. In reference tocancers or other unwanted cell proliferation, an effective amountcomprises an amount sufficient to cause a tumor to shrink and/or todecrease the growth rate of the tumor (such as to suppress tumor growth)or to prevent or delay other unwanted cell proliferation.

In some embodiments, an effective amount is an amount sufficient todelay development. In some embodiments, an effective amount is an amountsufficient to prevent or delay occurrence and/or recurrence. Aneffective amount can be administered in one or more doses. In the caseof cancer, the effective amount of the drug or composition may: (i)reduce the number of cancer cells; (ii) reduce tumor size; (iii)inhibit, retard, slow to some extent and preferably stop cancer cellinfiltration into peripheral organs; (iv) inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; (v) inhibit tumor growth;(vi) prevent or delay occurrence and/or recurrence of tumor; and/or(vii) relieve to some extent one or more of the symptoms associated withthe cancer.

As used herein, “treat”, “treatment”, “treating”, and the like refer toacting upon a condition (e.g., tau protein aggregation) with an agent(e.g., MKT-077) to affect the condition by improving or altering it. Theimprovement or alteration may include an improvement in symptoms or analteration in the physiologic pathways associated with the condition.The aforementioned terms cover one or more treatments of a condition ina patient (e.g., a mammal, typically a human or non-human animal ofveterinary interest), and includes: (a) reducing the risk of occurrenceof the condition in a subject determined to be predisposed to thecondition but not yet diagnosed, (b) impeding the development of thecondition, and/or (c) relieving the condition, e.g., causing regressionof the condition and/or relieving one or more condition symptoms (e.g.,reducing tau protein levels, increasing cognitive abilities). As itpertains to cancer treatment, beneficial or desired clinical resultsinclude, but are not limited to, any one or more of the following:alleviation of one or more symptoms (such as tumor growth ormetastasis), diminishment of extent of cancer, stabilized (i.e., notworsening) state of cancer, preventing or delaying spread (e.g.,metastasis) of the cancer, preventing or delaying occurrence orrecurrence of cancer, delay or slowing of cancer progression,amelioration of the cancer state, remission (whether partial or total).

The term “treating cancer” or “treatment of cancer” refers toadministration to a mammal afflicted with a cancerous condition andrefers to an effect that alleviates the cancerous condition by killingthe cancerous cells, but also to an effect that results in theinhibition of growth and/or metastasis of the cancer.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range.

Study

The current study evaluates the formulation and characterization of MKTencapsulated by a brain-targeted vector, such as PEG-PLGA NPs viananoprecipitation, coated in a sufficient amount (e.g., 2% w/v) of GSHto obtain greater BBB permeation and decrease toxicity in the targetarea or drug delivery (i.e., brain). PLGA NPs are FDA approved andwidely used in drug delivery due to their biodegradability into thebiocompatible compounds, lactic and glycolic acid, endocytosis throughclathrin-mediated pathways, and low toxicity [15]. PEG conjugation tothe PLGA polymer increases bioavailability of the NPs in blood due togreater solubility and low uptake by the reticuloendothelial system(RES) [16].

This formulation is contemplated to be a non-toxic alternative to reducepathological tau protein aggregation associated with AD and othertauopathies. Average nanoparticle size was found to be about275.73±12.41 nm, suitable for intravenous administration and brainpermeation, and have polydispersity index of about 0.066±0.051. Theencapsulation efficiency of the MKT NPs was about 25%. The nanoparticlesshowed steady, sustained release of MKT in in vitro settings. TRANSWELLin vitro BBB model permeation studies showed the permeation ofnanoparticles across the TRANSWELL model to be greater than drugsolution over 48 hours. As such, the formulation developed and describedherein has shown promise and potential efficacy as a potential therapyagainst AD and other tauopathies. The targeting system for MKT could bean effective sustained release treatment for AD and other relatedneurological disorders.

GSH was used to coat the NPs to induce trans-BBB permeability. GSH issynthesized in cells from amino acid precursors and acts in reducingoxidative stress to neural tissue. In addition, GSH transporters areabundantly found at the BBB interface and assist in the transport ofvarious compounds across the BBB [17]. A number of GSH-coated NPdelivery systems have been reported by the current inventors for thebrain-targeted delivery of the AD drug, Methylene blue [18], theParkinson's drug, tempol [19], and various cancer drugs such aspaclitaxel [20], doxorubicin [21], docetaxel [14], and methylprednisolone [22]. Furthermore, in vivo studies of 6-coumarin loaded GSHPLGA NPs injected peritoneally in C57BL/6 mice revealed higherconcentration of 6-coumarin in brain tissue as opposed to 6-coumarinuncoated PLGA NPs [20].

To study trans-BBB permeation of MKT NPs, in particular, however, thepresent study utilizes TRANSWELL permeable inserts for establishing anin vitro BBB model. TRANSWELL permeable inserts allow for the seeding ofmultiple cell lines on the apical and basolateral sides of the insertand sharing of media, thereby being considered the “gold standard” fortrans-membrane studies [23]. The model was established using rat brainendothelial (RBE4) and rat astrocytic (C6) cells; the interactions ofastrocytes with endothelial cells promotes the expression ofBBB-specific tight junctions and transport proteins in endothelial cells[14, 24].

The present study formulates and characterizes MKT NPs and alsodemonstrates brain-targeted delivery of MKT NPs as a potential treatmentagainst AD and other tauopathies. Cellular studies in AD models showthat the GSH-coated MKT NPs (MKT Glu-NPs) are effective in reducing tauprotein levels. TRANSWELL in vitro BBB permeation studies show that MKTGlu-NPs were also able to cross the BBB better than MKT drug solutionalone.

These newly generated MKT-NPs showed BBB permeability and tau reductionin experimental models. This embodiment of the formulation can be seenin FIG. 1. Although it can be seen in this embodiment that the PEG endgroups may be longer than GSH, which could shield the interactionbetween the GSH and its transporters, the PEG group does not coverentire surface area of nanoparticles. There is sufficient surface areaavailable for interaction with the BBB transporters. Moreover, PEG isvery hydrophilic which will help the interaction of hydrophilic GSH withtransporters present on the BBB. Higher BBB permeation has been shown byGSU coating on liposome with PEG end group [22]. The NPs show promise asa potential therapeutic option against AD and other tauopathies.

Materials and Methods

Materials

PEG-PLGA (5050 DLG, mPEG 5000) was synthesized and purchased fromLAKESHORE BIOMATERIALS (Birmingham, Ala.). MKT was purchased fromSIGMA-ALDRICH (St. Louis, Mo.). Reduced GSH was purchased from FISHERBIOREAGENTS (THERMO FISHER SCIENTIFIC Inc., Pittsburgh, Pa.). Phosphatebuffered saline (PBS) was purchased from CELLGRO (CORNING Inc., Corning,N.Y.). Acetone and methanol were both purchased from SIGMA ALDRICH (St.Louis, Mo.).

Preparation of GSH-Coated PEG-PLGA NPs

NPs encapsulating MKT were formulated by known methodologies, such asthe nanoprecipitation method according to a protocol previously reportedby the current inventors [18]. Briefly, 3.5 mg of MKT was dissolved in100 μL of methanol and added dropwise to 4 mL of acetone. 120 mg ofPEG-PLGA was subsequently added to the acetone and the solution wasvortexed for the proper dissolution of all components. The resultingsolution was added dropwise to 8 mL of deionized H₂O (stirring at 300rpm). The acetone was allowed to completely evaporate overnight. Theresulting NP solution was centrifuged at 4500×g for 35 minutes tocollect the NPs. The resulting supernatant was decanted to discard anyunencapsulated drug and free polymer and replaced with 10 mL of freshdeionized H₂O. GSH was coated onto the NPs according to a methodpreviously reported by the current inventors [20]. 20 mg of GSH wasadded to 1 mL of the NPs solution to achieve a 2% w/v GSH coating andwas allowed to incubate at room temperature for 30 minutes prior to use.

Characterization of NPs Formulations

The blank NPs, MKT NPs, and MKT Glu-NPs were characterized for physicalparameters. Particle size and polydispersity index (PDI) were analyzedthrough dynamic light scattering (DLS) using the DYNAMIPRO PLATE READER(WYATT TECHNOLOGY, CA). Samples were diluted to fit instrumentalspecifications.

Scanning Electron Microscopy (SEM) Analysis of NPs Formulations

SEM was utilized to investigate the physical integrity of blank NPs, MKTNPs, and MKT Glu-NPs. JOEL JSM-6490LV (JOEL INDUSTRIES, Tokyo, Japan)was used to visualize the samples. The samples were diluted according toinstrumental specifications and were loaded onto aluminum cylinderscoated with an adhesive carbon polymer. NPs formulations were viewed in65,000× magnification. 4 kV acceleration voltage was used to visualizethe blank NPs and MKT NPs formulations and 8 kV acceleration voltage wasused to visualize the MKT Glu-NPs.

Determination of Entrapment Efficiency

The methanol method was employed to determine the entrapment efficiencyof MKT by the NPs [20]. 1 mL of MKT NPs was ultracentrifuged for 5minutes at 12,000 rpm to separate the NPs from solution. The resultingsupernatant was carefully removed and replaced with 1 mL of coldmethanol and placed in 4° C. refrigerator overnight to allow for acomplete extraction of MKT from the NPs. The methanol supernatant wasread by UV spectroscopy (COLE PARMER, Vernon Hills, Ill.) at awavelength of 492 nm (λ_(max)) and compared to standard dilutions of MKTdrug in methanol (r²=0.9934). The entrapment efficiency was calculatedby using the following formula:

${{entrapment}\mspace{14mu}{efficiency}} = {\frac{{actual}\mspace{14mu}{drug}\mspace{14mu}{yield}}{{theoretical}\mspace{14mu}{drug}\mspace{14mu}{yield}} \times 100\%}$

In Vitro Drug Release

The drug release profile of MKT from MKT NPs and MKT Glu-NPs wasinvestigated using a method previously described by the currentinventors in [14]. 0.05 mg equivalent of MKT aqueous solution, MKT NPs,and MKT Glu-NPs were placed in dialysis membrane tubing (MWCO 10,000kDa) and placed in 20 mL of release medium comprised of PBS (pH 7.4)stirring at 100 rpm and 37° C. 1 mL aliquots were removed atpredetermined intervals over 7 days and analyzed by UV spectroscopy atwavelength of 492 nm (λ_(max)) and compared to UV absorbance of standardaqueous dilutions of MKT drug (r²=0.9943).

Cell Culture

BBB model: C6 (rat astrocytoma) cells were purchased from ATCC(CCL-107), and RBE4 (rat brain endothelial) cells were obtained viagift. Cell culture plates and flasks were purchased from CORNING Inc.(Corning, N.Y.). TRANSWELL permeable inserts were purchased from CORNINGInc. (No. 3460; Corning, N.Y.). Ham's F10 and MEM media and PBS solutionwere purchased from CELLGRO (CORNING Inc., Corning, N.Y.). 1% penicillinand streptomycin solution and fetal bovine serum (FBS) were purchasedfrom INVITROGEN (THERMO FISHER SCIENTIFIC Inc., Pittsburgh, Pa.). Rattail collagen I and human recombinant diluted basic fibroblast growthfactor were purchased from BD BIOSCIENCES (San Jose, Calif.).

Cell Culture, Treatment and Immunoblotting

AD model: HeLa cells stably expressing human tau were maintained inOPTIMEM media as described earlier [18]. Treatment with nanoparticles orcontrol were performed for 24 hours and samples analyzed by westernblotting, as described earlier [18].

Trans-Endothelial Electrical Resistance (TEER) of TRANSWELL BBB Model

The TEER of the BBB model was studied to investigate the expression ofBBB-specific tight junctions in the TRANSWELL model according to amethod previously reported by the current inventors [14]. TEER valueswere measured at the beginning and end of the BBB permeation study.Media was aspirated from the inserts and replaced with PBS before takingTEER measurements. STX2 electrode and EVOM² epithelial voltohmmeter(WORLD PRECISION Instruments, Sarasota, Fla.) were used to measure TEER.

It has been shown that TEER values can be used investigate in vitro theexpression of BBB-specific tight junctions ([18], [21], [29]-[31]).

In Vitro BBB Permeation Model

A TRANSWELL in vitro BBB model was established to study the BBBpermeation of MKT NPs and MKT Glu-NPs compared to drug solutionaccording to a method previously described by the current inventors in[18]. TRANSWELL permeable inserts were treated with 0.1% rat tailcollagen I on the apical and basolateral sides. After 24 hours, C6 (ratastrocytoma) cells were seeded onto the basolateral side of thepermeable insert (5×10⁴ cells). After 48 hours, RBE4 (rat brainendothelial) cells were seeded onto the apical side of the permeable(5×10⁴ cells). Cells were bathed in 1:1 mixture of Ham's F10:MEM mediasupplemented with 10% FBS with 1.5 mL in the basolateral chamber and 0.5mL in the apical chamber, and were allowed to incubate (37° C. and 5%CO₂) for 24 hours until confluence was established.

After 24 hours, media was replaced with a 1:1 mixture of Ham's F10:MEMmedia supplemented with 1% FBS (experimental media), and cells weretreated with 10 μM equivalent treatments of MKT aqueous drug solution,MKT NPs, and MKT Glu-NPs. 1 mL media samples were aliquoted from thebasolateral chamber at predetermined intervals over a 48-hour period toinvestigate the trans-BBB permeation of the treatments. Aliquots wereread by UV spectroscopy (at wavelength of 492 nm (λ_(max)) and comparedto UV absorbance of standard dilutions of MKT drug in experimental media(r²=0.9990).

Results

Characterization of NPs Formulations

Three different samples of NPs formulations were characterized forphysically-relevant parameters. Size and PDI were investigated throughDLS; data is presented in Table 1. Blanks NPs were found to have size of244.37±14.45 nm and PDI of 0.0498±0.395; MKT NPs were found to have sizeof 275.73±12.41 nm and PDI of 0.0657±0.0508; and MKT Glu-NPs were foundto have size of 230.17±8.02 nm and PDI of 0.0302±0.0152. Samples wereread in triplicate (n=3) and reported as mean values±SD. The MKT NPswere significantly larger than blank NPs (p<0.05).

TABLE 1 Data from physical characterization of NPs formulations via DLS.Samples were read in triplicate (n = 3) and reported as mean value ± SD.Size (nm) PDI Blank NPs 244.37 ± 14.45 .0498 ± .0395 MKT NPS 275.73 ±12.41 .0657 ± .0508 MKT Glu-NPs 230.17 ± 8.02  .0302 ± .0152

Scanning Electron Microscopy (SEM) Analysis of NPs Formulations

JOEL JSM-6490LV (JOEL Industries, Tokyo, Japan) SEM was used tovisualize the morphology of blank NPs (FIG. 2A), MKT NPs (FIG. 2B), andMKT Glu-NPs formulations (FIG. 2C). Surface analysis of NP formulationsshowed that physical integrity was maintained by all samples. Size andsize distribution of respective NP formulations visualized through SEMcorroborated with data obtained via DLS.

Determination of Entrapment Efficiency

The efficiency of MKT encapsulation by the MKT NPs formulation wasinvestigated through the methanol method. UV spectroscopy analysisrevealed the encapsulation efficiency to be 29.98±1.37%. The valuesyielded by the encapsulation efficiency study were used to determine themolarity of the NPs solution and select the proper volume of NPformulations utilized in cell treatments. Data was obtained intriplicate (n=3) and reported as mean encapsulation efficiency±SD.

In Vitro Drug Release

In vitro release studies were carried out to investigate the rate ofdrug release by the NP formulations. Three (3) samples werestudied—aqueous MKT solution, MKT NPs, and MKT Glu-NPs—over 7 days.Approximately 100% of the drug was released by the aqueous solutionwithin the initial 4 hours of the study; approximately 100% of the drugwas released by the MKT Glu-NPs in 72 hours (FIG. 3); and approximately25% of the drug was released from the MKT uncoated NPs at the end of the7-day study (FIG. 3). The aqueous drug solution showed a rapid releaseof the drug, and both NP formulations investigated showed a sustainedrelease of the drug from the NP polymer due to slower degradation of thepolymer matrix of NPs. All samples showed initial burst release of thedrug. Samples were read in triplicate (n=3) and reported as mean percentdrug release±SD.

Trans-Endothelial Electrical Resistance (TEER) of TRANSWELL BBB Model

TEER values of the TRANSWELL in vitro BBB model are presented in Table2. TEER values were read for inserts of all treatment groups (i.e., drugsolution, MKT NPs, MKT Glu-NPs) before treatment at 0 hours and aftertreatment course was completed at 48 hours. TEER values weresignificantly higher after treatment for all experimental groups thanbefore treatment (p<0.05). TEER values were read in quadruplicates (n=4)and reported as mean resistance (Ωcm²)±SD.

TABLE 2 TEER values for all treatment groups taken before and aftertreatment. TEER values were measured with STX2 electrode and EVOM²epithelial voltohmmeter (WORLD PRECISION Instruments, Sarasota, FL) andread in quadruplicates (n = 4) reported as mean Ω cm² ± SD. Beforetreatment (Ω cm²) After treatment (Ω cm²) Drug solution 251.44 ± 17.48294.56 ± 18.15 MKT NPs 301.84 ± 54.52 386.70 ± 80.23 MKT Glu-NPs 271.41± 14.18 354.29 ± 24.44

In Vitro BBB Permeation Model

TRANSWELL permeable inserts were used to establish an in vitro BBB modelto investigate the trans-BBB permeability potential of equivalentconcentrations of NP formulations as compared to aqueous drug solutionover a 48-hour study period. At the end of the 48-hour study period, 20%of the drug solution permeated, 3% of the MKT NPs permeated through(FIG. 4A), and 72% of the MKT Glu-NPs permeated through the BBB model(FIG. 4B). The permeation profiles of the drug solution and MKT Glu-NPswere relatively similar for the first 24 hours of the study. MKT Glu-NPsdrastically increased trans-BBB permeation between 24-48 hours whilepermeation of drug solution was relatively unchanged. Treatments werecarried out in quadruplicates (n=4) and reported as mean percentage drugpermeated±SD.

Effect of MKT NPs on Tau Level in a Cellular AD Model

In order to determine whether the newly formulated MKT-NPs are active, acellular AD HeLa cell model that stably expresses human tau was used.Cells were treated with 3 μM of MKT uncoated NPs or MKT Glu-NPS or drugin solution or various controls for 24 hours. Western blot analysis ofsamples showed that treatment with MKT Glu-NPs causes a similarreduction in tau levels as MKT in solution (FIGS. 5A-5B), though asdiscussed MKT in solution cannot permeate the BBB effectively, as MKTGlu-NPs can. Further, uncoated MKT NPs showed slightly lower reductionin tau compared to MKT in solution (FIGS. 5A-5B). Overall, cellular datasuggests MKT Glu-NPs are functionally active and effective to reducingtau levels.

Discussion

The NPs were formulated according to the nanoprecipitation method, whichemploys the use of two distinct phases of formulation: oil phase andwater phase. The oil phase is comprised of the NP vector and drug, andthe water phase is that from which the oil phase solvent evaporates tocause the precipitation of the NP formulations. NPs developed by thismethod exhibit spherical structures and maintain physical integrity andproper morphology suitable for intravitreal injection and drug delivery.Coating of GSH onto the NPs does not compromise the physical integrityof the NPs formulations. The GSH exists on the outer layer of the NPformulation and is thought to coat the NP polymer through electrostaticinteractions between the side chains of the GSH molecule and chargedfunctional groups of the PEG component of the PEG-PLGA vector [14].

FIG. 1 depicts a representation of the MKT Glu-NP formulations. The drugMKT is encapsulated in the hollow sphere created by the self-assembly ofhydrophobic NP vector lipid chains in water phase upon the evaporationof the oil phase solvent.

The sizes of the NPs formulations were over 200 nm. The sizes of the MKTNPs were significantly greater than that of the blank NPs, whichsuggests that the increase in MKT NPs size can be attributed to thepresence of drug molecule in the NPs. Furthermore, the smaller size ofthe MKT Glu-NPs as compared to the MKT NPs may be attributed to the GSHcoating of the NPs, which may induce a more compact shape due to thevery hydrophilic nature of GSH [20]. In addition, the lower sizes of theMKT Glu-NPs may also be attributed to displacement of any MKT existingon the surface of the NPs by GSH [18]. The sizes of NPs in eachformulation were normally distributed as suggested by PDI data.

Contrarily, monodisperse formulations have greater particle sizefluctuations which may induce excessive aggregation in solution [18].SEM data confirmed the uniform size distribution of the NPsformulations, as the SEM micrographs of NPs showed spherical morphologywith fine particles. The GSH coating of the MKT Glu-NPs can be confirmedby the reflective coat of the formulations as observed in the SEM data.The size data obtained from the MKT Glu-NP formulations suggest that theformulation is suitable for intravenous delivery and BBB permeation[25].

An issue present in the formulation of NPs encapsulating hydrophilicdrugs is their rapid escape into the water phase without properencapsulation by the NPs [26]. The cause of this complication is thelowered encapsulation efficiency as compared to that of hydrophobicdrugs. However, the encapsulation efficiency of nearly 30% seen in thepresent study is adequate for any future in vivo experimentation due toappropriate and physiologically relevant amount of MKT present in theNPs formulations. Any alteration in the NP formulation method throughchanges in amounts of drug and polymer chosen would likely lower theencapsulation efficiency of derived NP formulations.

The NP formulations exhibited sustained release of MKT from the polymer.MKT NP and MKT Glu-NP formulations had an initial burst release profile,which is the release of unencapsulated drug that exists on the surfaceof the NPs. The drug solution exhibited a rapid release of the drug inthe initial 4 hours of investigation that remained consistent over theremainder of the study. The MKT Glu-NPs exhibited sustained release ofthe drug over 72 hours until complete release was achieved. MKT NPs wereable to release approximately 20% of the drug by the end of the study.The delayed release of the MKT NPs could be due to the slowerdegradation of the polymer matrix of PEG-PLGA. Drug release from NPpolymers occurs mainly through diffusion and swelling-induced lysing ofthe NP matrix. In addition, the long chains of the PEG-PLGA polymerinduce the attraction of water molecules to the NP matrix to enter theNPs and release drug through hydration [27]. It is assumed that therapid release of drug from the MKT Glu-NPs could be induced by greaterattraction of water by the hydrophilic GSH-coated surface of the MKTGlu-NPs which would lead to greater hydration and release of drug fromthe NPs matrix. It is reasonable to assume that drug release profiles ofMKT from MKT Glu-NP formulations may differ in in vivo situations due tothe complex interactions of the NPs with biological compounds.

Proper BBB physiology is maintained by the adequate expression of tightjunctions in brain endothelial cells through interactions withassociated neuroglial cells to block the permeation of potentially toxicsubstances into neural tissue [28]. TEER values measure the endothelialresistance of the TRANSWELL model which, in turn, determines whether theappropriate tight junctional barrier is maintained by the BBB model forthe proper replication of physiological phenomenon [23]. TEER valuesobtained from the TRANSWELL model in the current study ranged from about231.84 Ωcm² to about 313.6 Ωcm² prior to treatment with drug solutionand NP formulations and from about 271.04 Ωcm² to about 356.16 Ωcm²following treatment. The TEER values were significantly increased at theend of the 48-hour study period suggesting the increased expression oftight junctions in endothelial cells throughout the study period. Thereported TEER values in the present study are higher than thosepreviously reported for mono-, co-, and tri-cultures of humanmicrovascular endothelial cells with human astrocyte and pericyte cellsgrown on TRANSWELL permeable inserts similar to the methodologydescribed herein [23], suggesting that the TRANSWELL in vitro BBB modelin the current study is a physiologically-relevant indicator oftrans-BBB permeation of drug compounds.

Treatments with drug solution, MKT NPs, and MKT Glu-NPs in the TRANSWELLBBB model revealed that the MKT Glu-NPs permeate through the BBB atgreater rates than both drug solution and MKT NPs. It is reasonable tobelieve that the greater rate of MKT Glu-NP permeation is due to theendocytosis of MKT Glu-NPs by the endothelial cells to allow passagethrough the BBB. In addition, it was surprising to find that the MKTGlu-NPs showed a drastic increase in BBB permeation between 24-48 hoursof the study period despite the increased expression of tight junctionalproteins in endothelial cells of the TRANSWELL model, as evidenced bythe significantly greater TEER values after the study was completed. Inother words, despite the increased expression of tight junctionalproteins, which would typically lead to less permeation through the BBB,MKT Glu-NPs were still capable of effectively permeating the BBB.

Functionality of newly generated MKT-NPs was tested in awell-characterized cellular model of AD and tauopathies as describedpreviously [18]. As seen in FIGS. 5A-5B, treatment with MKT Glu-NPs andMKT-solution showed greater and comparable reduction in tau levels.However, the uncoated MKT-NP formulation showed slightly lower reductionin tau level (FIGS. 5A-5B), which can be explained by the fact that MKTNPs had lower permeation than both the drug solution and MKT Glu-NPs,due to the blockage of lipid-compound permeation through the BBB. Thecurrent inventors have previously reported the trans-BBB permeation of anumber of GSH-coated NP formulations [14, 18, 20, 21] and have reportedthe permeation of GSH-coated NPs in C57BL/6 mouse model when injectedperitoneally [20]. Similar BBB permeation results can be expected inanimal models of AD when injected with MKT Glu-NPs of MKT.

CONCLUSION

The present study formulated and characterized NPs encapsulating MKT andenabled the brain targeting of the NPs formulations. The NPs weresuccessfully formulated with biologically compatible vector and particlesize as well as uniform size distribution. GSH was successfully coatedonto the surface of the NPs, and induced greater and more sustained drugrelease profile in solution as compared to drug solution and MKT NPs.The MKT Glu-NPs were shown to permeate a physiologically-relevant invitro model of the BBB better than the drug solution and MKT NPs. MKTGlu-NPs showed functional and similar reduction in tau levels, like MKTin solution, in a cellular model of AD and tauopathies. Taken together,the data enables targeting of the brain with the described MKT Glu-NPformulation for the reduction of tau-related pathology in models of AD.

It is further expected that the MKT Glu-NP formulation would beeffective in providing anti-cancer effects in a subject or patient,based on [32], of which a current co-inventor is a co-author, where MKTwas seen to have anti-cancer effects.

REFERENCES

-   1. Kolarova, M., et al., Structure and pathology of tau protein in    Alzheimer disease. International journal of Alzheimer's    disease, 2012. 2012.-   2. Jeganathan, S., et al., The Natively Unfolded Character of Tau    and Its Aggregation to Alzheimer-like Paired Helical Filaments†.    Biochemistry, 2008. 47(40): p. 10526-10539.-   3. Friedhoff, P., et al., Structure of tau protein and assembly into    paired helical filaments. Biochimica et Biophysica Acta    (BBA)-Molecular Basis of Disease, 2000. 1502(1): p. 122-132.-   4. Hyman, B. T., J. C. Augustinack, and M. Ingelsson,    Transcriptional and conformational changes of the tau molecule in    Alzheimer's disease. Biochimica et Biophysica Acta (BBA)-Molecular    Basis of Disease, 2005. 1739(2): p. 150-157.-   5. Garcia-Sierra, F., et al., Conformational changes and truncation    of tau protein during tangle evolution in Alzheimer's disease.    Journal of Alzheimer's Disease, 2003. 5(2): p. 65-77.-   6. Maccioni, R. B., J. P. Munoz, and L. Barbeito, The molecular    bases of Alzheimer's disease and other neurodegenerative disorders.    Archives of medical research, 2001. 32(5): p. 367-381.-   7. Thies, W. and L. Bleiler, 2013 Alzheimer's disease facts and    figures. Alzheimer's & dementia: the journal of the Alzheimer's    Association, 2013. 9(2): p. 208-245.-   8. Miyata, Y., et al., Synthesis and initial evaluation of YM-08, a    blood-brain barrier permeable derivative of the heat shock protein    70 (Hsp70) inhibitor MKT, which reduces tau levels. ACS chemical    neuroscience, 2013. 4(6): p. 930-939.-   9. Propper, D., et al., Phase I trial of the selective mitochondrial    toxin MKT 077 in chemoresistant solid tumours. Annals of    oncology, 1999. 10(8): p. 923-927.-   10. Koren III, J., et al., Rhodacyanine derivative selectively    targets cancer cells and overcomes tamoxifen resistance. PloS    one, 2012. 7(4): p. e35566.-   11. Jinwal, U. K., et al., Hsc70 rapidly engages tau after    microtubule destabilization. Journal of biological chemistry, 2010.    285(22): p. 16798-16805.-   12. Weisberg, E. L., et al., In vivo administration of MKT causes    partial yet reversible impairment of mitochondrial function. Cancer    research, 1996. 56(3): p. 551-555.-   13. Tatsuta, N., et al., Pharmacokinetic analysis and antitumor    efficacy of MKT, a novel antitumor agent. Cancer chemotherapy and    pharmacology, 1999. 43(4): p. 295-301.-   14. Grover, A. H., Anjali; Pathak, Yashwant; Sutariya, Vijaykumar,    Brain-Targeted Delivery of Docetaxel by Glutathione-Coated    Nanoparticles for Brain Cancer. AAPS PharmSciTech, [In Press.    Accepted 28th May, 2014].-   15. Danhier, F., et al., PLGA-based nanoparticles: an overview of    biomedical applications. Journal of controlled release, 2012.    161(2): p. 505-522.-   16. Wacker, M., Nanocarriers for intravenous injection—The long hard    road to the market. International journal of pharmaceutics, 2013.    457(1): p. 50-62.-   17. Valdovinos-Flores, C. and M. E. Gonsebatt, The role of amino    acid transporters in GSH synthesis in the blood-brain barrier and    central nervous system. Neurochemistry international, 2012.    61(3): p. 405-414.-   18. Jinwal, U. K., et al., Preparation and Characterization of    Methylene blue Nanoparticles for Alzheimer's disease and other    Tauopathies. Curr Drug Deliv, 2013.-   19. Carroll, R. T., et al., Brain-targeted delivery of Tempol-loaded    nanoparticles for neurological disorders. Journal of drug    targeting, 2010. 18(9): p. 665-674.-   20. Geldenhuys, W., et al., Brain-targeted delivery of paclitaxel    using glutathione-coated nanoparticles for brain cancers. Journal of    drug targeting, 2011. 19(9): p. 837-845.-   21. Geldenhuys, W., et al., Brain-targeted delivery of doxorubicin    using glutathione-coated nanoparticles for brain cancers.    Pharmaceutical development and technology, 2014(0): p. 1-10.-   22. Lee D H, Rotger C, Appeldoorn C C, et al. Glutathione PEGylated    liposomal methylprednisolone (2B3-201) attenuates CNS inflammation    and degeneration in murine myelin oligodendrocyte glycoprotein    induced experimental autoimmune encephalomyelitis. J Neuroimmunol    2014; 274(1-2): 96-101.-   23. Hatherell, K., et al., Development of a three-dimensional,    all-human<i> in vitro</i> model of the blood-brain barrier using    mono-, co-, and tri-cultivation TRANSWELL models. Journal of    neuroscience methods, 2011. 199(2): p. 223-229.-   24. Garcia, C. M., et al., Endothelial cell-astrocyte interactions    and TGFβ are required for induction of blood-neural barrier    properties. Developmental brain research, 2004. 152(1): p. 25-38.-   25. Hatakeyama, H., et al., Factors governing the in vivo tissue    uptake of transferrin-coupled polyethylene glycol liposomes in vivo.    International journal of pharmaceutics, 2004. 281(1): p. 25-33.-   26. Bilati, U., E. Allémann, and E. Doelker, Development of a    nanoprecipitation method intended for the entrapment of hydrophilic    drugs into nanoparticles. European Journal of Pharmaceutical    Sciences, 2005. 24(1): p. 67-75.-   27. Wischke, C. and S. P. Schwendeman, Principles of encapsulating    hydrophobic drugs in PLA/PLGA microparticles. International Journal    of pharmaceutics, 2008. 364(2): p. 298-327.-   28. Cecchelli, R., et al., Modelling of the blood-brain barrier in    drug discovery and development. Nature Reviews Drug Discovery, 2007.    6(8): p. 650-661.-   29. Ghaffarian, R., Muro, S. Models and Methods to Evaluate    Transport of Drug Delivery Systems Across Cellular Barriers. I Vis.    Exp. (80), e50638, doi:10.3791/50638 (2013).-   30. Etame, A. B., Smith, C. A., Chan, W. C. W., Rutka, J. T. Design    and Potential Application of PEGylated Gold Nanoparticles with    Size-Dependent Permeation Through Brain Microvasculature,    Nanomedicine: Nanotechnology, Biology, and Medicine, 2011, 7,    992-1000.-   31. Garberg, P., Ball, M., Borg, N., Cecchelli, R., Fenart, L.,    Hurst R. D., Lindmark, T., Mabondzo, A., Nilsson, J. E., Raub, T.    J., Stanimirovic, D., Terasaki, T., Oberg, J. O., Osterberg, T. In    Vitro Models for the Blood-Brain Barrier. Toxicol in Vitro, 2005,    19, 299-334.-   32. Koren J III et al. (2012) Rhodacyanine Derivative Selectively    Targets Cancer Cells and Overcomes Tamoxifen Resistance. PLoS ONE    7(4): e35566. doi:10.1371/journal.pone.0035566.-   33. Paul R. Lockmana et al., Brain uptake of thiamine-coated    nanoparticles, Journal of Controlled Release 93 (2003) 271-282.

All referenced publications are incorporated herein by reference intheir entirety. Furthermore, where a definition or use of a term in areference, which is incorporated by reference herein, is inconsistent orcontrary to the definition of that term provided herein, the definitionof that term provided herein applies and the definition of that term inthe reference does not apply.

The advantages set forth above, and those made apparent from theforegoing description, are efficiently attained. Since certain changesmay be made in the above construction without departing from the scopeof the invention, it is intended that all matters contained in theforegoing description or shown in the accompanying drawings shall beinterpreted as illustrative and not in a limiting sense.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention that, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A method of treating a tauopathy in a subject,comprising administering a therapeutically effective amount of acomposition comprising nanoparticles to the subject having thetauopathy, wherein the nanoparticles comprise: MKT-077 encapsulated by abiocompatible, non-toxic polymer, coated by glutathione, and a pluralityof poly(ethylene glycol) end groups around a surface of eachnanoparticle, and wherein a hydrophilic nature of said glutathionepermits said MKT-077 to effectively permeate across a blood-brainbarrier of the subject having the tauopathy.
 2. The method of claim 1,wherein said biocompatible, non-toxic polymer includes poly(ethyleneglycol)ylated poly-(lactide-co-glycolide) to encapsulate said MKT-077.3. The method of claim 1, wherein said nanoparticles are coated in 2%w/v of said glutathione.
 4. The method of claim 1, wherein upon saidadministration of said composition to said subject, said MKT-077 has asustained release from said nanoparticles of over about 72 hours.
 5. Themethod of claim 4, wherein about 100% of said MKT-077 is released fromsaid nanoparticles over said sustained release.
 6. The method of claim1, wherein said composition has a size of less than about 300 nm.
 7. Themethod of claim 1, wherein said plurality of poly(ethylene glycol) endgroups extend further away from said surface of said each nanoparticlethan said glutathione disposed on said surface of said each nanoparticlein order to provide a surface area of said plurality of poly(ethyleneglycol) end groups to interact with transporters present on saidblood-brain barrier.
 8. The method of claim 1, wherein the tauopathy isAlzheimer's disease.
 9. The method of claim 1, wherein saidbiocompatible, non-toxic polymer comprises polylactic acid (PLA). 10.The method of claim 1, wherein said biocompatible, non-toxic polymercomprises polycaprolactone (PCL).